Controlled hydration or swelling of cartilage provides a means of determining functional properties of cartilage and of other extracellular matrices. Specifically, we have used controlled hydration of cartilage to measure important physical/chemical properties of the collagen network and of the proteoglycans (PG) independently within the extracellular matrix. This approach entailed modeling the cartilage tissue matrix as a composite material consisting of two distinct phases: a collagen network and a concentrated proteoglycan solution trapped within it; applying various known levels of equilibrium osmotic stress; and using physical-chemical principles and additional experiments to determine a "pressure-volume" relationship for both the PG and collagen phases independently. In pilot studies, we used this approach to determine pressure-volume curves for the collagen network and PG phases in native and in trypsin-treated normal human cartilage specimen, as well as in cartilage specimen from osteoarthritic (OA) joints. In both normal and trypsin-treated specimen, collagen network stiffness appeared unchanged, whereas in the OA specimen, collagen network stiffness decreased. Our findings highlighted the role of the collagen network in limiting normal cartilage hydration, and in ensuring a high PG concentration in the matrix, both of which are essential for effective load bearing in cartilage and lubrication, but are lost in OA. These data also suggest that the loss of collagen network stiffness, and not the loss or modification of PGs may be the incipient event leading to the subsequent disintegration of cartilage observed in OA.[unreadable] [unreadable] One shortcoming of this approach, however, was that it required a significant amount of tissue to obtain the osmotic titration curves. This lead to long equilibration times requiring person-days to study a single cartilage specimen, making this approach unsuitable for routine pathological analysis or for use in tissue engineering applications. Recently, we developed a new micro-osmometer to perform these experiments in a practical and rapid manner. This instrument can measure minute amounts of water absorbed by small tissue samples (< 1 microgram) as a function of the equilibrium activity (pressure) of the surrounding water vapor. A quartz crystal detects the water uptake of a specimen attached to its surface. The high sensitivity of its resonance frequency to small changes in the amount of adsorbed water allows us to measure the mass uptake of the tissue specimen precisely. Varying the equilibrium vapor pressure surrounding the specimen induces controlled changes in the osmotic pressure of the tissue layer. To validate the methodology, we used synthetic polymer gels with known osmotic properties. To illustrate the applicability of the new apparatus, we measured the swelling pressure of tissue-engineered cartilage specimen.[unreadable] [unreadable] The micro-osmometer will eventually permit us to obtain a profile of the osmotic compressibility or stiffness of multiple cartilage specimens simultaneously as a function of depth from the articular surface to the bone interface. It will also allow us to quantify the contributions of individual components of ECM (such as aggrecan, hyaluronic acid and collagen) to the total osmotic pressure. Moreover, it should allow us to assess the osmotic compatibility and mechanical integrity of developing tissues and of tissue-engineered cartilage (or ECM) with the hope of improving integration following implantation.[unreadable] [unreadable] Recently, we have developed an atomic force microscopy (AFM) technique for mapping the local elastic and viscoelastic properties of tissues. Many of the impediments that have previously hindered the use of the AFM in high-throughput probing of inhomogeneous samples, particularly biological tissues, have been addressed. The technique utilizes the precise scanning capabilities of a commercial AFM to generate large volumes of compliance data and automatically extracts the relevant elastic properties from the data. In conjunction with results obtained from micro-osmometry and biochemical analysis, this technique will allow us to map spatial variations in the load-bearing capacity of cartilage specimens.